Biological product and process of obtaining same



-- 1" "UNITED STATE Patented Mar. 20, 1945 7 OBTAINING SAME l BIOLOGICAL rnonUc'r AND PROCESS or Norbert H. Fell, Detroit, Mich, assignor to Parke. Davis & Company, Detroit, Mich., a corporation of Michigan "13 Claims.

The invention relates to preparation of products useful in preventing the allergic symtoms to which many people are subject as the result of their sensitivity to specific sensitizing substances; The invention relates .more particularly to a product which can be artificially or synthetically produced and which is useful for developing either active or passive resistance or immunity in sensitized individuals.

I have found that I can produce, what to all intents and purposes appears to be, both active and passive immunity against. histamine and similar deleterious substances which are present in the animal or human'body during, or at about the same time as, the manifestations of the symptoms of anaphylaxis and other allergic reactions. whereby it is possible for sensitive individuals to avoid allergic reactions, regardless of the type Of allergens to which they are sensitive.

In carrying out the invention, I combine histamine or a like substance, as before mentioned.

with a large molecule or compound of high molecular weight in a haptenic linkage, or a haptenelike linkage. to form a complex which can be used as an antigen for developing a. resistance, a type of refractoriness. or active immunity in the body of a sensitized individual, or for injecting into animals. from which later an antiserum can be obtained containing an apparent antibody for neutralization. of the effects of. histamine or deleterioushistamine-like substances.

My invention makes possible the type of treatment of allergic conditions which is non-specific and does not depend upon the recognition of and protection against individual allergens which may produce symptoms in any given case.

A valuable feature of the products of this invention is that they are effective when administered orally as well as when given by injection. For example. the antigen products described in the examples hereinaftergiven canbe used directly to prevent allergic manifestations or can be administered to the animal organism and an antiserum later obtained from the organism by the usual methods knownjor obtaining antiserums. The antiserums can then be used either orally or by injection.

Since th new products of my invention function for all practical purposes like true antigens wccntaining histamine or histamine-like compounds combined in what appears to be a true as'to how the products ofthe invention carry out theirfunctions but only by the'description of the invention given herein and as defined by the appended claims. I

This application is a continuation-in-part of my United States application, Serial No. 225,612, filed August 18, 1938.

The histamine or like deleterious substance which I combine with a large molecule is in most cases an imidazole compound. In order to combine the histamine or histamine-like compound with the protein or similar large molecule, a derivative of an imldazole may first be produced in which at least one of the atoms of the imidazole ring is substituted by a radical containing a chemical group capable of coupling with a protein. Such final products are therefore of the type, I

where H represents a substituted or unsubstituted imidazole radical and A represents a divalent residue joined by a valence to the group H and by another valence to a protein molecule, P.

The residue A is a coupling component and can be present in a variety of forms or types of chemical groups. The substituted or unsubstituted imidazole radical can first be combined by means of a suitable'imidazole derivative with the coupling component, A, to give the combination,

' I-IA. If a group is present in A of this combibecause it is generally more convenient to couple nation which is capable of coupling with a protein, then H-A is directly coupled with the protein, P. If no such is present, then A of H-A is modified so that coupling will occur.

- Instead of combining Hand A before reacting with the protein, the coupling component, A, can

first be coupled with the protein and thereafter combined with the 'imidazole. However, the preferred method is the first one above mentioned,

the protein last.

Various protein products may be used for the coupling, but the preferred substances are those proteins which are foreign to the animal or human organism for which the antigens are intended to be used. After the antigen is prepared. it can be used or injected directly for developing active immunity in the bodyof the sensitized individual, or, the antigen can first be injected into animals which are bled after a suitable period of time and the serum obtained from the blood of the animals then used, with or without further purification or fractionation of the serum, to neutralize the effects of histamine or histamine-like substances responsible for the manifestations of allergy. v

The invention may be illustrated by the following examples:

and allowedto stand for about minutes.

Example 1-Preparation of 0. nitro benzamide intermediate Two holes of histamine base are dissolved in hot dry chloroform and a solution of one mole of p-nitro benzoyl chloride dissolved in ether is stirred in. A heavy precipitate forms immediately which has a waxy appearance. The supernatant liquid layer is removed from the precipitate and centrifuged to remove all insoluble ma-v terial. The precipitate is dissolved in boiling water, filtered while hot from any material which fails to dissolve, and the filtrate cooled to' give crystals of the imidazolyl ethyl p nitr'o benz-- amide. The crystals can be washed with acetone and ether and then dried at 110 C. They melt at 204 C. The compound prepared by this example can be represented by the formula;

An analysis of the crystalline product gives the theoretical amount of nitrogen for a compound of this formula.

water added. The solution is then adjusted to a pH of 8.1-8.2 with 10% sodium hydroxide solution. I This alkaline horse serum globulin solution is chilled in an ice bath. ,A'solution of a diazonium salt of imidazolyl ethyl p-amino benzamide, prepared for instance as described in Ex-- amples l, 2 and 3 and starting with about 830 mgms. of histamine base, is added slowly to the chilled horseserum globulin solution. More 10 1. NaOH is added until the mixture turns a bloocie red color. Any insoluble material present at this I stage can be centrifuged off and discarded. An

excess of hydrochloric acid is added to the supernatant liquid until it becomes acid to Congo red paper. This acidification causes a heavy flocculent precipitate to come out. The precipitate is centrifuged off. The centrifuged precipitate is washed with acidified saline and then dissolved in excess dilute alkali hydroxide to give a clear blood-red solution. To this solution,0.5

' phenol is added and then a few drops of con' centrated HCl to bring the pH to about 1.6,

4 after which the solution is put through a Mandler Example 2Preparation of an amino-beazamide I compound One-half gram of imidazolyl ethyl p-nitro be'nzamide, prepared as in Example 1, is added to 50 cc. of a hot solution of 3.5 grams of ferrous sulfate (FeSO4.7H2O). 35 cc. of a 2.5% solution of ammonium hydroxide are then gradually added to the ferrous, sulfate solution while heating on the steam bath. After adding all the ammonium hydroxide solution, the reaction mixture is heated for 30 minutes and the ferric hydroxide filtered oif and the filtrate set aside to cool. The filtrate contains the'imidazolyl-ethyl p-amino bcnzamide. This compound may be obtained in solid form by allowing the filtrate to stand whereupon spontaneous crystallization sometimes occurs, or, evaporating on the solvent,

' or adding an organic solvent in which the amino benzamide is insoluble, thereby obtaining a precipitate or crystals which can be filtered oil. The crystals have a melting-point of 189-l9l C. and differ from the corresponding p-nitro compound in being very soluble in acid and very insoluble in alkali. The product of this example is represented by the formula,

HN N

c 1'; b Example 3Preparation of diazo compound of imidazolyl ethyl p-amino benzamide The filtrate from Example 2 containing the imidazolyl ethyl p-amino benzamide is neutralized with hydrochloric acid and 173 mgms. of

sodium nitrite added while chilling the reaction mixture in an ice-salt mixture. The reaction mixture is acidified with 4.5 cc. of normal HCl It contains the diazotlzed imidazolyl ethyl p-amino benzamide.

Example 4 Preparation of a histamine-ozoprotein antilren Five grams of horse serum globulin are taken up in cc. of cold water and 50 cc. of boiling shown to contain antibodies against the complex antigen and against histamine. Such an immune serum. injected into guinea-pigs sensitized to horse serum provides them with protection (passive immunity) against subsequent anaphylactic shock from horse serum. l

Human subjects may also be treated in the same manner as animals to give them an active or a passive immunity which enables them to avoid the uncomfortable and deleterious symp-v toms accompanying allergic reactions.

Instead of using horse serum globulin in this example, I may use some other protein, such as egg alubumin or large organic molecules capable of coupling with a histamine-diam compound or a like combination of histamine, or histamine-like .compound, with a diazotlzedamino compound.

bination capable of reduction to give a free amino Instead of using nitro benzoyl chloride, other nitro organic acid halides may be used to comblue with the histamine to form an amide comgroup for purposes of diazotization,

As already mentioned above, various other proteins maybe combined with other H--A type compounds. This is illustrated by the following examples, such as'Exampl'e 5 below wherein purifled cow serum pseudo globulin is coupled with a diazonium salt of imidazolyl' ethyl p-amino benzamide, and Example 6 below, in which a protein is lin are prepared by precipitating cow serum with coupled with chloracetyl histamine.

Example 5.p-Imidazolyl ethyl (p-diazo proteo) benaamide 10 grams of purified cow serum pseudo globu- 13% sodium sulfate, discarding the precipitate and increasing the concentration of sodium sul-. fate to 24% and collecting the precipitate formed at the latter concentration. The precipitate is dialyzed, to remove inorganic salts, down to the equivalent of 0.8% NaCl. The dialyzed pseudo globulin is then put'through a Mandler filter and the total nitrogen content determined by ter.

lution. This alkaline cow serum pseudo globulin solution is chilled and a solution of a diazonium salt of imidazolyl ethyl-p-amino benzamide, prepared for instance as described in Examplesl, 2 and 3 and starting with about 1650 grams of histamine base, is added slowly. More NaOHsolution is added until the mixture turns a blood-red color. Any insoluble material which appears at this point can be separated or filtered of! and thrown away. Excess hydrochloric acid i added to the filtrate or supernatant liquid until it becomes ac'd to Congo red paper. This acidification causesa heavy fiozculent precipitate to come out. The precipitate is centrifuged off. The centrifuged precipitate is washed with acidified saline and then dissolved in excess dilute alkali hydroxide to give a clear blood-red solution. To this solution, 0.5% phenol i added and then a few drops of concentrated HCl to bring the pH to about 7.6, after which the solution is put through a Mandler fil- The solution is ampouled and can be submitted to sterility tests before using it for injections.

Example 6.Chloracet ul histamine antigen The essential reactions of this example may be represented as follows: v

I N H (Chloroacetyl histamine l II the "temperature is raised to and the gases and excess SOClz are distilled on. The remaining liquid is cooled and filtered. It is chloracetyl chlo- "ride of boiling point 103 C.

(2) 500 rngfihistamine are dissolved iulO to.

hot chloroform and 1 cc. 1.5 g) of chl-nl'acetyl chloride areadded drop by drop. The so utinn becomes cloudy and a gummy precipitate form It is allowed to stand /2 hr; and is then extracted with 10 cc. of water. The aqueous solution is evaporated to dryness andextracted with ether. The ether extract is dried in a desiccator until the gummy material becomes crystalline. The yield is 576 mg. of pure chloracetyl histamine analyzing approximately the theoretical percentage oi. nitrogen.

(3) 2 cc. of scarlet fever antitoxin mxed with. v

3 cc. of physiological saline and 5 cc. of phosphate bufler (pH=8), are'cooled on ice, and 2 cc. of a solution of 576 mg. of chloracetyl histamine in This product, and also that of Example 5, is useful for the same purposes as the product of Example 4. It can be evaporated at low temperature and pressure to give a dry product capable of being dissolved in water again and used.

Example 7Histamine isocgjano antigen The chief reactions ,of this example may be represented as follows:

'(1) 222 mg. histamine are dissolved in 50c. ethyl alcohol and 1 cc. chloroform and 10 cc. alcoholic KOH are added. The solution is warmed ,hr. at 50. It is then evaporated on the steam bath until salts crystallize out. These are extracted with absolute alcohol and the extract evaporated nearly to dryness and chilled. A heavy oily material forms. This is dried to a solid substance over P205. Analysis shows almost the theoretical percentage of nitrogen for histamine isonitrile.

160 mg. ofoxide of mercury are added. The sus-.

pension lsheat-ed at 110? for 20 minutes on an oil bath. It is -cooled, filtered and the filtrate evaporated to dryness. The dry solid product is histamine isocyanate.

(3) 2 cc. of scarlet fever antitoxin, 3 cc. of physiological saline and 5 cc. of phosphate bufler pH 8) are cooled on ice, and 100 mg. of histamine isocyanate are added. The solution is stirred and allowed to stand for hr. It is precipitated with 10% trichloracetlc acid, centrifuged, washed, redissolved in dilute alkaline solution and the pH adjusted to 7.2. isocyano protein antigen and is suitable for injection or use orally for combatting the efl'ects I of allergy.

Example 8-Histamine isocyano protein antigen.

I (Histamine isocyanate) 500 mg. of histamine are dissolved in cc. of

warm chloroform and 10 cc. of a phosgene solution in toluene are added. The mixture is heated for hour and the chloroform and toluene distilled oil. The solution remaining is cooled, filtered and the precipitate of histamine isocyanate thereby obtained is dried.

The dry isocyanate is reacted with protein to obtain the final antigen in the manner described above under Example 7 and the antigen is useful for the same purpose asthat' of Example 7.

Example -9-Preparation of 2- (azobenzidine azo proteo-) histamine The essential transformation in this example may be illustrated as follows:

(a) n-.-co-cmonmm H-N N Z-(azobenzidine azo pretco-) histamine It is a solution of histamine 1) 1 gram of benzidine is dissolved in 200 ml.

,color by the mixture.

of concentrated hydrochloric acid and the mixture chilled to 0-5 C. Gold normal sodium nitrite solution is added to the chilled benzidine solution until starch iodide paper is turned a blue A solution ofdiazo benzidine chloride is thus obtained.

(2) A quantity of 0.6 gram of histamine dissolved in ml. of water is chilled and poured slowly into the diazobenzidine chloride solution and the reaction mixture allowed to stand for 15 minutes. 250 ml. of a cold solution of 3 grams of sodium acetate dissolved in 500 ml. of water and 20 m1. of normal potassium carbonate solution are then added and the'solution allowed to stand for 15 minutes longer. -The product-in this step is the result of a coupling reaction with histamine. This can be proved by coupling the product of step 2 with a-naphthol instead of a protein and analyzing the a-naphthol coupled product for nitrogen. For example, 475 ml. of the cold solution containing the reaction product of histamine and azobenzidine diazonium chloride can be reacted with 0.65 gram of a-naphthol. The a-naphthol can be added while stirring in an ice bath and the reaction mixture allowed tostand hour. The blue-black precipitate which forms is filtered off, washed, dried and analyzed for nitrogen content. It analyze approximately the theoretical (21.6% nitrogen) for 2-(azobenzidine azo-a-naphthol) -histamine of the formula, C27H15ON7.

(3) At the end of the 15 minutes, 100 ml. of

the reaction mixture are added to a chilled solution (40 ml.) of scarlet fever antitoxin diluted 1 to 5 and made alkaline with normal potassium carbonate solution. The reaction mixture is then allowed to stand onice for /2 hour. The 2- (azobenzidine azo proteo-) histamine in the product is precipitated twice with 10% trichloracetic acid, dissolved again in alkali and phenol preservativeadded up to alooncentration of 0.5%. The product is then put through a sterilizing Mandler filter and the hydrogen ion concentration of the filtrate adjusted to pH 7.15. This product is suitable ior injection as described for the other products described in the preceding examples.

The scarlet fever antitoxin used in step 3 can be replaced by other proteins and especially other proteins foreign to the animal body to which the antigen is to be administered. Other large molecules, especially high molecular weight organic compounds, can be coupled to the histamine and its derivatives in a like manner to form similar products.

Example Ill-Reaction product of imidazolvl ethyl isocyano acetamide and'protein The chemical transformations of this example may be represented as follows, 1 I 1 n-c=c-'-cmoaiNm-| cicmcoc1 N NH 1 CHC I =C-CHICHI-NH-OO-CH1NEC v NEW NH I! The chloracetyl histamine of the first step can be obtained for example by the procedure given for the first two steps of Example 6.

The chloracetyl histamine is reacted with ammonia, for example in the form of an excess of concentrated aqueous ammonium hydroxide solution. The resulting amino acetamlde derivative of histamine is extracted with a solvent, such as ether, and the solvent evaporated. The amino acetamide derivative is reacted with potassium hydroxide and chloroform and then with mercuric oxide in a manner analogous to that given for histamine in Example '1. or with phosgene in a manner analogous to that given under Example 8. The resulting product is imidazolyl ethyl iso- This product is finally reacted with a large organic molecule such as a protein to yield an antigenie product.

Example 11Reaction product of S-(p-methuluminoethub)-1-imidaayl-p-benzene diazonium v A chloride and a protein The chemical transformations of this example may be represented as follows:

B-(B-methylene iminoethyD- l-imidazyl-p-nitrobenzene p-IS-Q-methylaminoethyl)-l-imidazyl] bonzene-diazonium chloride l I Protein [P]- N N- s O N I l H l L MH,

In the first step, the histamine can be dissolved in dilute acetic acid and formalin added, after which the mixture is made alkaline with sodium hydroxide solution and heated. The sol- -vent is removed from the reaction mixture by evaporation and the dry residue extracted with ether. The ether is evaporated from the extract,

leaving behind'the formaldehyde histamine.

The second step is carried out by heating a mixture of the formaldehyde histamine and pchloronitrobenzene, if necessary in the presence of a solvent.

The S-(p-methylene iminoethyl) -1-imidazyl-pnitrobenzer'ie is separated in the usual manner from any solvent used and then taken up in alcohol and treated with hydrogen under pressure in the presence of a paladium catalyst for reduction of the nitro and the methyleneimino groups. The resulting -(p-methylaminoethyl)? l-imidazyl-p-aminobenzene is filtered from the catalyst, the alcohol is evaporated ofi and the solid product taken up in a solution of strong hydrochloric acid, cooled, and a solution of sodium nitrite added slowly while'cooling. When the diazotization is complete, there is added to the solution of the p-[ 5-(B-methylaminoethyl)- l imidazyl] benzene-diazonium chloride a solution of serum globulin made alkaline with alkali carbonate solution. After the cooled mixture stands for an hour it is precipitated with an agent used for precipitating proteins, such as trichloracetic acid, and then taken up in alkaline solution. It can be filtered through a Mandler filter to sterilize it and its pH adjusted to about 7.1 when it is ready for use.

EwampZe.12--Reaction product of protein and p- [fl- 5-imidazyl) -ethylcarbamyl] -benzoic acid The steps of this example are as follows: n-c=c-cn.cnmm

E Step 1 l p-lfl-(imidazyDethylcarbamyH- benzonitrile Step 2 K01! and then acid Protein [P] Step 3 (1) 400 mg. p-cyano benzoyl chloride are dissolved in 15 cc. dry CHClz. 368 mg. histamine in CHCla are added. A white precipitate forms immed iately. This is taken upin 10 cc..methanol (hot) and filtered with 2 vols. of cold water. A white precipitate comes down. It is washed with ether and dried. The dry product is p-cyano benzoyl histamine'or p-[fl-(imidazyl) ethylcar-. bamyllbenzonitrile and melts at 194-195". Its nitrogen content is 21.74% against a theoretical value of 21.87%.

(2) mg. of p-cyano benzoyl histamine are added to 5 cc. of 1.0% potassium hydroxide on a steam bath and gradually dissolved. It is neutralized and v evaporated to dryness with methanol, dissolved in pyridine and concentrated. The oily residue is dried over calcium chloride to a. solid or melting point Bil-65 C. It is paid-(5- imidazyl) ethylcarbamyl] benzoic acid.

(3) The benzoic acid derivative of step 2 is mixed with an excess of thionyl chloride and allowed to stand for an hour or two, after which the unreacted thionyl chloride is distilled off.

under reduced pressure. The residue is the acid chloride and is mixed andreacted with an equivalent quantity of analkaline solution of protein as in the previous examples. The reaction product of theprotein with the p[B-(5 'imidazyl-ethylcarbamyl] benz oyl chloride can be directly used or can be dialyzed, preferably after adjusting to the desired pH, and filtered through a sterilizing filter before use.

, Histidine Ho==c-cmcH-c 0 on Stepy Carbobenzoxy hist idine chloride yields the known compound, carbobenzoxy histidine, of melting point about 209 C. The latter compound is then converted in step 2 to its acid chloride by the usual method for converting a carboxylic acid to its acid chloride The de-carbobenzoxy antigen which results is by means of phosphorus pentachloride. The acid chloride is reacted in step 3 with the protein inslightly alkaline solution to give a useful combination with protein. However, if desired, one

can treat the protein reaction product with hydrogen in presence of palladium to produce reductive scisslon of the carbobenzoxy group, as indicated for step 4. In this case the final product is a protein derivative of histidine or car-boxy histamine.

Example 14-React ion product of S-(p-carbobenzoxy-amidoethyl) -1 -chloracetyl imidazole' with a protein The transformations of this example can be indicated as follows; (l) HO==T CHICHQ NHI O NH ol-i z-d-o Err-O H-C =c-crriom-uH-c -0 on.

Stop 2 N N-o-omon -(fl-corbobenzoxyamidoethyl)-l-chloracetyl imidatolo Step 3 Protein [P] I H Antigen Simpler derived antigen In the first step the new compound, 5- (p-carbobenzoxyamidoethyl) -imidazole, melting at about 190 C., can be obtained by cooling a solution of histamine in dilute alkaline solution and stirring the carbobenzoxy chloride into the solution. After a few hours the solution is extracted with ether, the ether evapcrated off of the extract and the residue cooled to give a white crystalline product. The residue can be recrystallized from suitable solvents if desired.

| Step1 H In step 2 the chloracetyl-chl'oride is reacted also an eflective antigen forprotecting against histamine and like products.

' Example 15-471mm: containing a sulfanilamide 9 m" The transformations of this example may be illustrated as follows: H-C==I=C-CHQCHQNBI N0:

N NE

n-c-c-cmonmn-soO N /NE M. pt. 180 0.

Ferrous sultate reduction m-Sulianilyl histamine Dlazotiza'tion IGINO, and H01) NsCi . H-C Ml-CHICHrNH- 5 Dr- 'N\ [NH Diuotiued m-sulianilyl histamine 1 Protein, [P]

N=N-lP] n-c-=c -o mcnr-NH- s o V Coupled antigen grams of m-nitro benzene sulfoohloride are dissolved in dry chloroform and 110 grams of histamine added slowly to the solution. The waxy precipitate which forms is filtered off and then dissolved in methyl alcohol. Ether is added to the methyl alcohol solution toreprecipitate the reaction product which is filtered off. It is pure crystalline N-(m-nitrobenzene sulfonyl) hista mine and is a new compound of melting point approximately C. V

'35 grams or the N-(m-nitro benzene sulfonyl) histamine are suspended in about 800 or 900 cc. of water and the suspension added to 2000 cc. of

a hot solution of 1'72 grams of ferrous sulfate,

FeSO4-7I-I2O'. The mixture is boiled 15 minutes and filtered. The filtrate is adjusted to a pH of 7.8 and placed in an ice box whereupon crystals separate out and are then filtered off and dried. The crystals consist of N-(m-amino benzene ulfonyl) histamine or m-sulfanilyl histamine hich has a melting point of 265 .C. with some charting.

The m-sulfanilyl histamine can be diazotized' by the usual procedure for dlazotizing primary aryl amines which comprises reacting it with an acidic solution of nitrous acid. After the diazotized m-sulfanilyl histamine is separated out of the reaction mixture, it can be coupled with a large organic molecule, such as a protein, in the manner described for other examples given above. The product is an antigen of exceptional value.

Example '16 Prepa ration of an antigen from, the

imidazolyl ethyl p-amino benzamide intermediate of Example 2 The reactions of this example are represented schematically by the following,

Partially 88 all l (e. g. with l ofl l Diazotiae I II a v =o c mcmaua-c-O-mm lCouplvwith lprutvin [1) Antigen 20.0 grams of imidazolyl ethyl p-amino benzamide, obtained for instance as described under Example 2, having a melting point of approximately 191 C., are dissolved in several hundred cc. of acetic anhydride and warmed for an hour. The reaction solution is cooled with ice and the crystals which separate are filtered of! and thoraavaocc oughly dried. They melt at about 92 C. and are substantially pure imidazolyl ethyl p-acetamin'o benzamide.

The acetamino derivative is dissolved in water containing alkali sufficient to make the solution group can be hydrolyzed oil. of the acetamido compound. The resulting fi-(l-carbobenzoxy imidazolyl) -p-amino-benzamide can be extracted with an organic solvent from the alkaline reaction solution and recovered, for example by evaporation of the solvent or concentrating and cooling. It is then diazotized and coupled with a protein, as described in other examples given above.

in order to obtain an antigenic product.

If desired, the protein-coupled antigen of this example can be converted into a simpler antigen by reductive scission of the carbobenzoxy group,

as described above under Example 14. The prod-' uct thus obtained is the same as that of Examples 4 and 5, when the protein of each or those examples is used.

From the foregoing description of the invention it will be apparent that, in its broader aspects, the invention embodies many different combinations heretofore unknown and also includes numerous intermediate products required for the preparation or the same which are distinctly new in themselves. y

In the claims appended hereto theexpression animal body may, in its broader sense, include the human body as well as the bodies 01 animals.

What I claim is:

1. Method for obtaining a product efiective against deleterious compounds of histamine-like properties present in the body at the time of an allergic manifestation which comprises coupling a protein with diazotized imidazolyl ethyl p-amino benzamide.

2. The coupled reaction product of amammalian 7 serum protein and a derivative of histamine in which the fi-(fi-imidazolyl) ethyl radical of histamine is attached by way of a nitrogen atom to an organic radical comprising a radical ofthe class consisting of isocyanate, rat-halogenated 'acyl, diazonium salt andcarboxy halide radicals.

3. The coupled reaction product of a protein and a derivative of histamine in which the ,B-(5- imidazolyl) ethyl radical of histamine is attached by way of a nitrogen atom to an organic radical comprising. a radical of the class consisting of isocyanate, (ii-halogenated acyl, diazonium salt and carboxy halide radicals.

4. The coupled reaction product of a horse serum globulin and a derivative of histamine in which the B-(5-imidazolyl) ethyl radical of histamine is attached. by way of a nitrogen atom to an organic radical comprising a radical of the class consisting of isocyanate, zit-halogenated acyl,

diazonium salt and ,carboxy halide radicals.

5. The coupled reaction product of a protein and a diazotized primary amino-substituted carboxylic acid amide of histamine.

. The coupled reaction product of a protein The reaction liquid as'raoco and a diazotised p-imidazolyl ethyl p-amino benzamide. i

' '1. The coupled reaction product of a mam malian serum protein and a diasotized primary amino-substituted carboxylic acid amide o1 histamine.

8. 'Ihe coupled reaction product of a mammalian serum protein and a diazotized p-imidazolyl ethyl p-amino benzamide.

9. The coupled reaction product of a horse globulin and a diazotized p-imldazolyl ethyl p-aminobenzamide.

10. Process which comprises coupling a protein with a derivative of histamine in which the s-(5-imidazo1yl) ethyl radical of histamine is attached by way of a nitrogen atom to an organic radical comprising a radical of the class consisting of isocyanate, run-halogenated acyl, diozonium salt and cal-boxy halide radicals.

Patent No. 2,572,066.

.GERTIFICAi'E OF CORRECTION.

11. Process which comprises coupling a mammalian serum with a derivative 01 histamine in which the fi-(ii-lmidaaolyl) ethyl radical of histamine it attached by way of a. nitrogen atom to an organic radical comprising a radical of the class consisting of isocyanate, a-haiogen- 'Matedacyl, diasonium salt and car-boxy halide NORBERT H. FELL.

moron 2o 19i 5.

NORBERT H. FELL.

It is hereby certified that error appears-in the printed specification of theabove numbered patent requiring correction as follows: Page 2, first column, line 55, Example 2,' for that portion of the formula reading "N0 read --NH read -is attached--';

page 9, second column, line 14., claim 11, for "it attached" and that the said Letters Patent should be read with this correction therein that the same may conform to the record of the case in the Patent Office.

Signed and sealed this 29th (so a1) d of January, A. D.19 +6.

Leslie Frazer First Assistant Conrnis'sioner of Patents.

as'raoco and a diazotised p-imidazolyl ethyl p-amino benzamide. i

' '1. The coupled reaction product of a mam malian serum protein and a diasotized primary amino-substituted carboxylic acid amide o1 histamine.

8. 'Ihe coupled reaction product of a mammalian serum protein and a diazotized p-imidazolyl ethyl p-amino benzamide.

9. The coupled reaction product of a horse globulin and a diazotized p-imldazolyl ethyl p-aminobenzamide.

10. Process which comprises coupling a protein with a derivative of histamine in which the s-(5-imidazo1yl) ethyl radical of histamine is attached by way of a nitrogen atom to an organic radical comprising a radical of the class consisting of isocyanate, run-halogenated acyl, diozonium salt and cal-boxy halide radicals.

Patent No. 2,572,066.

.GERTIFICAi'E OF CORRECTION.

11. Process which comprises coupling a mammalian serum with a derivative 01 histamine in which the fi-(ii-lmidaaolyl) ethyl radical of histamine it attached by way of a. nitrogen atom to an organic radical comprising a radical of the class consisting of isocyanate, a-haiogen- 'Matedacyl, diasonium salt and car-boxy halide NORBERT H. FELL.

moron 2o 19i 5.

NORBERT H. FELL.

It is hereby certified that error appears-in the printed specification of theabove numbered patent requiring correction as follows: Page 2, first column, line 55, Example 2,' for that portion of the formula reading "N0 read --NH read -is attached--';

page 9, second column, line 14., claim 11, for "it attached" and that the said Letters Patent should be read with this correction therein that the same may conform to the record of the case in the Patent Office.

Signed and sealed this 29th (so a1) d of January, A. D.19 +6.

Leslie Frazer First Assistant Conrnis'sioner of Patents. 

